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The following message was posted to: PharmPK
I am currently conducting some experiments to try to determine
hepatocyte concentrations of a drug and metabolite. I recently tried a
suspended hepatocyte method in which at designated time points, I took a
sample of media containing hepatocytes, added it to a tube containing 3M
KOH and silicone/mineral oil and centrifuged. The supernatant was
representative of media and the KOH (bottom layer) contained the cells.
When I assayed for drug and metabolite via LC-MS/MS, the metabolite (and
less so the drug) peak areas were junky for the KOH/cell samples, but
looked good for the media samples. Is anyone aware of another method
that I could employ to modify or replace the KOH?
Melanie S. Joy, Pharm.D., FCCP
Associate Professor
UNC School of Medicine
Division of Nephrology and Hypertension
UNC Kidney Center
CB #7155, 7005 Burnett Womack Building
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The following message was posted to: PharmPK
Is your compound stable in base? 3M KOH is capable of destroying a
lot of
compounds Laos how did you get rid of the KOH before LC-MS/MS?
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Melanie,
what is the logD/P of your compound, it will help to design a
methodology that can assist your analytical endeavors using organic
extraction that will terminate the reaction, yet preserve the
integrity of the cells. With LCMS, the use of strong ions and buffers
will inevitable complicate the analytical profile. Using polar
gradients with sampel divert times will help clean up your profile.
There are many optins, just depends on what the characteristics of the
molecule and your ultime analyte(s) is/are.
1.Using an isoamylalcohol and ether extraction methodology may help to
differentiate polar from non polar.
2.Methylene chloride extractions can help also (modifiers can be used
to change the polarity of the mixture).
3. Rapid colling with centrifugation ina microfuge tube of the
suspension sample aliquot followed by differential extraction can also
be used.
3. Have you thought of using an isolated perfused model in rats and
mice to determine metabolic disposition?
4. Qualyst's system of hepatocytes?
5. Do you know what you're looking for on teh basis of the parent
molecular structure?
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